Screening of Infection-related Factors from Salmonella Pullorum by in Vivo Induced Technology, Identification of Plasmid pSPI12 and Function Analysis of IpaJ Protein
|Keywords||Pullorum Salmonella pSPI12 ipaJ In vivo induced antigen technology Infection related factor Real-time PCR|
Pullorum Salmonella multi-infringement within the 20-day-old chicks, causing white diarrhea, high mortality rate. Adult chicken infection does not appear typical clinical symptoms, but long-term carrier state. Adult chicken spleen and reproductive tract, the bacteria can survive for more than 40 weeks. Sexual maturity period, the bacteria can invade the ovaries and fallopian tubes, vertical transmission to the next generation. Due to the strain energy level and vertical transmission, can cause great harm to the poultry industry. At present, in the Western developed countries, the disease has been eliminated or basically eliminated, however, still occur in individual small flocks, still more pullorum outbreak of domestic flocks. The causative gene for pullorum Salmonella is of great significance for the understanding of its pathogenesis and effective prevention and treatment of pullorum. In this study, IVIAT (in vivo induced antigen technology) screening and identification of the pullorum Salmonella infection-related factors, for a comprehensive understanding of pullorum Salmonella pathogenesis provide a foundation. Salmonella virulence plasmid in bacteria pathogenic process plays an important role in the study from the pullorum Salmonella Isolation and identification of a new plasmid pSPI12 was only virulence-related genes in the plasmid ipaJ for understanding of early bacterial infection mechanism provides a favorable material. 1 pullorum Salmonella S06004, genomic expression library of one of the main pathogens of poultry, pullorum Salmonella pathogenesis is still not very clear. The the experiment IVIAT (in vivo antigen-induced technology) Filter pullorum Salmonella infection in vivo factor in order to understand the gene expression of the bacteria in the body. First, we must build pullorum Salmonella genomic expression library. S06004 genome digested with Sau3AI, recycling-4 kb of size in the 0.1 kb fragment. Digested with BamHI series of prokaryotic expression vector (pET30a, b, c), recovered the linear vector fragment was dephosphorylated. After the digestion of the genomic fragment connected by appropriate ratio and the prokaryotic expression vector, transforming Escherichia coli DH5, pick transformants, plasmid was extracted after liquid culture, and analysis of the inserted fragment size distribution and the rate of the insertion of the fragments using vector specific primers . Then extracted from the plate plasmid and transformed into E. coli BL21 (DE3), S06004 genome was constructed prokaryotic expression library. To use IVIAT technology screening of Salmonella pullorum infection-related factor to obtain the ten infected pullorum Salmonella positive serum antigen-binding expression in the sub-step S06004 vitro cultures mixed state to remove the corresponding antibodies, while taking advantage of the indirect ELISA for detection of the adsorption effect. Then after the adsorption treatment serum S06004 genomic expression library immunization screening, primary screening and secondary screening, 45 positive clones were screened out. Positive clones were sequenced after analysis of the protein sequences contained therein. Screened protein involved in biological macromolecules synthesis and metabolism, transport proteins, regulatory factors, energy metabolism associated protein of unknown function protein, reflecting the bacteria in the course of infection in the body, in addition to the expression of virulence genes, bacterial expression associated protein maintain normal growth and metabolism and response body changing in vivo environment, provides a basis to reveal the in vivo activity of the bacteria. Real-time quantitative PCR pullorum Salmonella S06004 infection related factors in vitro and in vivo expression differences according the IVIAT screened positive clone sequence and protein sequence homology than the results, design 11 pairs of S06004 infection-related factors (traV, gatD of PbPc, stbc, Deor, emrB, dapA, phoQ trkH adhE yhaN) the detection primer, with chicken Salmonella typhi GMK gene as an internal reference. Total RNA extract pullorum Salmonella S06004 vitro liquid LB medium, reverse transcribed into cDNA; to SPF chickens animal models, the use of intravenous cultured in vitro the S06004 bacterial immune, infected blood in different time periods, from the blood collected bacteria, total RNA was extracted, reverse transcribed into cDNA using quantitative PCR infectivity factor mRNA expression in vitro and in vivo culture difference. The results show that: the individual genes showed high expression levels phoQ; gene expression has continued to rise, such as gatD the; of Trav, dapA, Deor, emrB and trkH increased first and then decreased; adhE, PBPC, and STBC downward trend ; yhaN was down then up trend. Reflecting each infection-related factors have different levels of upregulation in vivo infection in different time mRNA levels are not consistent, the expression level with respect to in vitro culture. 4. Pullorum Salmonella pSPI12-plasmid isolation and sequence analysis using suppression subtractive hybridization method, constructed subtractive cDNA library pullorum Salmonella and Salmonella enteritidis, screened pullorum Salmonella-specific nucleotide sequence. The partial sequence splicing a the ipaJ gene with Salmonella choleraesuis the C500 the attenuated strain plasmid pSFD10 of the ipaJ highly homologous. In order to verify the the pullorum Salmonella ipaJ gene exists in a similar plasmid, the kanamycin resistance gene inserted strain S06004 ipaJ gene of Salmonella pullorum bacteria constructed insertion mutants SIM 12. Plasmids were extracted from the mutant strain, transformed E. coli DH5, kanamycin resistance positive clones, the plasmid was extracted from the obtained the Family ipaJ gene plasmid pSPI12 of the plasmid construct to sequencing of the pMD18T carrier analysis to obtain the complete sequence of the pSPI12 map. Homology analysis display pSPI12 and pSFD10 highly homologous, both contain a virulence-related genes ipaJ. PCR identification results show that our laboratory 105 pullorum Salmonella isolates containing the gene, In addition to Salmonella choleraesuis C500 vaccine strains, other Salmonella choleraesuis strains and other serotypes intestinal Salmonella subspecies not expand increase the target gene. Southern blot analysis showed that ipaJ gene is only present on the plasmid. pSFD10 is ColE plasmid F plasmid-mediated conjugal transfer, so we speculate choleraesuis the C500 Salmonella pSFD10 plasmid may be caused by Salmonella pullorum pSPI12 transfer part of the base mutation. 5 the pullorum Salmonella IpaJ, protein functional analysis of Shigella ipa responsible for encoding Invasin involved in the regulation of the process of the bacteria invade the cells. To explore of Salmonella pullorum IpaJ protein function, constructed the reply plasmid PCR carries ipaJ gene? The 2.1-ipaJ electroporated into ipaJ Mutant SIM 12 lines, and get a reply the strains SIM 12 (ipaJ,). Avian kidney epithelial cells (CKC) epithelial cell infection model, compared S06004 strains, the SIM 12 strains and the SIM12 (ipaJ) strains cell invasion and proliferation in the intracellular difference, the results show the invasive ability of the mutant strains, and in the extracellular within the proliferative capacity was significantly lower than the wild-type strain and reply strains. Parasitic spaces as its spleen macrophages Salmonella pullorum, the present study was to avian macrophage Department of HD-11 for macrophage infection model, the difference of Comparative three bacterial cell invasion and intracellular proliferation ability, the mutant invasion ability of strains are significantly lower than the wild-type strain and reply strains, but no significant difference in the proliferation trend in macrophages, increasing trend from infection to 5 hr, 5 hr after intracellular bacteria began to decline . In vivo analysis and comparison of wild strains, mutants and reply strain on the 10-day-old Highland white layers pathogenic mutant mortality compared with the wild-type strain reduced about 14 times, and reply to strain and the wild-type strain . The extraction of total RNA, using RT-PCR amplified from the infection of the S06004 total cellular RNA out ipaJ gene showed that this gene was expressed during infection from infected macrophages and spleen cells of chicken. Western-blot experiments show vitro the expression IpaJ protein can pullorum, Salmonella positive serum reaction, further Description IpaJ protein may be involved in the infection process of the Salmonella pullorum bacteria on the body, and mutations in this gene cause a reduction in bacterial virulence. In summary, this study IVIAT Screening pullorum Salmonella infection in vivo expression of infection-related factor, 45 protein function involves the synthesis and degradation of bacterial biological macromolecules, regulatory proteins, transport proteins, energy metabolism related protein, phage functions related protein of unknown function protein. Select a factor of 11 real-time PCR analysis results showed that in the course of infection in the body, the amount of expression of these genes in vitro culture status compared to have some degree of increase. Isolated from the strains of Salmonella pullorum a new plasmid pSPI12, for the 4080 bp in size. Sequence analysis revealed the presence of bacterial virulence genes ipaJ this plasmid and its encoded protein may be involved in bacterial infection early in the process of cell invasion. Animal experiments have shown the gene mutations ipaJ will lead to the reduction of bacterial virulence, so we speculate ipaJ gene is pullorum Salmonella virulence genes may. PCR analysis results show that the gene isolated in the laboratory to save time span of 40 years, 105 pullorum Salmonella in Salmonella choleraesuis vaccine strain C500 containing the gene and similar plasmid, not isolated the gene in other Salmonella choleraesuis strains and intestinal Salmonella subspecies strains.